Field of the Invention
The present invention relates to methods of degrading or hydrolyzing a polysaccharide, such as chitin or cellulose, comprising contacting said polysaccharide with an oxidohydrolytic enzyme, such as CBP21 or a GH61 protein, wherein said degradation or hydrolysis is carried out in the presence of at least one reducing agent and at least one divalent metal ion. The invention also extends to the use of additional saccharolytic enzymes such as hydrolases and beta-glucosidases to increase the level or extent of degradation and to fermentation of the resulting sugars to generate an organic substance such as an alcohol, preferably ethanol, which may be used as a biofuel.
Description of the Related Art
Efficient enzymatic conversion of crystalline polysaccharides is crucial for an economically and environmentally sustainable bioeconomy, but remains unfavourably inefficient.
The transition to a more environmental friendly economy has spurred research on enzymes capable of efficiently degrading recalcitrant carbohydrates, such as cellulose and chitin (FIG. 1A), for the production of biofuels (Himmel et al., 2007, Science 315: 804). Cellulose is the most abundant organic molecule on the earth and offers a renewable and seemingly inexhaustible feedstock for the production of fuels and chemicals. Chitin is a common constituent of fungal cell walls, shells of crustaceans and exoskeletons of insects. It is the second most abundant polymer in nature and each year more than one billion tons of chitin are produced in the biosphere, mainly by insects, fungi, crustaceans and other marine organisms. Chitin is abundantly available as a by-product from aquaculture, one of the fastest growing bioproduction industries on earth.
The conversion of cellulosic feedstocks into ethanol has the advantages of the ready availability of large amounts of feedstock, the desirability of avoiding burning or land filling the materials and the cleanness of the ethanol fuel. Wood, agricultural residues, herbaceous crops and municipal solid wastes have been considered as feedstocks for ethanol production. These materials primarily consist of cellulose, hemicellulose and the non-polysaccharide lignin. Once the cellulose is converted to glucose, the glucose is easily fermented by yeast into ethanol.
A variety of microorganisms exist for fermenting the products of hydrolysis of polysaccharides to yield desirable end products such as alcohol. Selection of appropriate microorganisms allows the products of hydrolysis of cellulose, chitin and other polysaccharides to be fermented to yield useful products, such as alcohol.
The predominant polysaccharide in the primary cell wall of biomass is cellulose, the second most abundant is hemi-cellulose and the third is pectin. The secondary cell wall, produced after the cell has stopped growing, also contains polysaccharides and is strengthened by polymeric lignin covalently cross-linked to hemicellulose. Cellulose is a homopolymer of anhydrocellobiose and thus a linear beta-(1-4)-D-glucan, while hemicelluloses include a variety of compounds, such as xylans, xyloglucans, arabinoxylans and mannans in complex branched structures with a spectrum of substituents. Although generally polymorphous, cellulose is found in plant tissue primarily as an insoluble crystalline matrix of parallel glucan chains. Hemicelluloses usually hydrogen bond to cellulose, as well as to other hemicelluloses, which help stabilize the cell wall matrix.
Bacteria and fungi have evolved complex enzymatic systems enabling their growth on plant material rich in cellulose, but these organisms typically require weeks, months or even years to decompose a fallen log or a tilled corn stalk. Likewise, microorganisms contain enzymatic systems for degrading chitin. Bacterial chitinase helps to provide a carbon source for bacterial growth. Insects produce chitinase to digest their cuticle at each molt. In plants, chitinase is thought to provide a protective role against parasitic fungi. For chemical or fuel production from these same cellulose- and chitin-containing materials, industry requires affordable chemical or enzymatic systems that can do the job in hours or in days.
Traditionally, enzyme systems capable of degrading such carbohydrates were considered to consist of two types of hydrolytic enzymes called glycoside hydrolases: endo-acting enzymes that cut randomly in the carbohydrate chain and processive exo-acting enzymes (chito- or cellobiohydrolases), which degrade the polymers from chain ends (FIG. 1B). Although this model is generally accepted, it remains difficult to understand how, e.g., an endoglucanase would be capable of pulling a single polysaccharide chain out of its crystalline environment and forcing the chain productively into its active site cleft (FIG. 1B).
Ever since cellulases caught the interest of biochemists, there have been speculations about the possible existence of a substrate-disrupting factor that could make the crystalline substrate more accessible to hydrolytic enzyme (Reese et al., 1950, J. Bacteriol. 59: 485). Recently, it was discovered that microorganisms that breakdown chitin indeed produce a protein that increases substrate accessibility and potentiates hydrolytic enzymes (Vaaje-Kolstad et al., 2005, J. Biol. Chem. 280: 28492; FIGS. 1C and D). The first example was a single-domain protein called CBP21 (CBP for Chitin-Binding Protein) produced by the chitinolytic bacterium Serratia marcescens. This protein has been classified as carbohydrate-binding module (CBM) and belongs to family CBM33 as defined by the CAZy nomenclature (Boraston et al., 2004, Biochem. J. 382: 769, Henrissat, 1991, Biochem. J. 280 (Pt 2): 309). Another example concerns two CBM33-containing proteins from Thermobifida fusca, called E7 and E8, which potentiate chitin hydrolysis by chitinase and cellulose hydrolysis by cellulases (Moser et al., 2008, Biotechnol. Bioeng. 100(6): 1066-77). Like CBP21, E7 is a single domain protein only comprising a CBM33 domain. E8 is a three-domain protein, meaning that it carries two domains in addition to a CBM33 domain.
It has recently been shown that proteins presently classified as family 61 glycoside hydrolases (GH61) in the CAZy nomenclature act synergistically with cellulases (Harris et al., 2010, Biochemistry 49: 3305) and are structurally similar to CBM33 proteins (Harris et al., 2010, supra; Karkehabadi et al., 2008, J. Mol. Biol. 383: 144; FIG. 1E). While CBM33 and GH61 have little sequence similarity, the structural similarity is evident (FIGS. 1D and 1E), including a diagnostic fully conserved arrangement of the N-terminal amino group, an N-terminal histidine and one other histidine residue (FIG. 1F; Karkehabadi et al., 2008, supra) forming a promiscuous metal-binding site (see below). On the basis of available literature data, including a recent comprehensive study of several GH61 proteins, it seems highly unlikely that GH61 proteins are endoglucanases, as originally thought (Harris et al., 2010, supra). Like CBM33 proteins, GH61 proteins do not have a substrate-binding cleft or pocket, nor do they possess a characteristic arrangement of acidic amino acids that could indicate a glycoside hydrolase activity. Instead, both types of proteins show an almost flat substrate-binding surface (FIGS. 1D and 1E; Harris et al., 2010, supra; Vaaje-Kolstad et al., 2005, J. Biol. Chem. 280: 11313). All in all, these results and observations show that CBM33 and GH61 proteins have similar functions, i.e., potentiating the efficacy of known hydrolytic enzymes (glycoside hydrolases) acting on crystalline polysaccharides. Furthermore, these results and observations strongly suggest that these proteins do so employing the same type of mechanism. So far, this mechanism has remained elusive.
The present invention provides methods of degrading or hydrolyzing a polysaccharide, such as chitin or cellulose, comprising contacting said polysaccharide with an oxidohydrolytic enzyme in the presence of at least one reducing agent and at least one divalent metal ion.